Tertiary lymphoid organs in wild boar exposed to a low-virulent isolate of African swine fever virus.

Despite the great interest in the development of a vaccine against African swine fever (ASF) in wild boar, the immunological mechanisms that induce animal protection are still unknown. For this purpose, tertiary lymphoid organs (TLOs) of wild boar were characterised and compared with mucosa-associated lymphoid tissues (MALTs) by histopathology, histomorphometry and immunohistochemistry (CD3, CD79, PAX5, LYVE1, fibronectin). In addition, real-time polymerase chain reaction (qPCR) and immunohistochemistry (p72) were used to evaluate the presence of ASF virus (ASFV) in blood and tissues samples, respectively. TLOs were observed in animals infected with a low-virulent ASFV isolate (LVI), animals co-infected with low and high-virulent ASFV isolates (LVI-HVI) and animals infected only with the high virulence isolate (HVI). TLOs in LVI and LVI-HVI groups were located adjacent to the mucosa and presented a similar structure to MALT. Immunoexpresion of p72 observed in the inflammatory cells adjacent to TLOs/MALTs confirmed its development and reactivity generated by ASF attenuated isolates. Immunohistochemical evaluation, based on cellular composition (T and B lymphocytes), and histomorphometrical study revealed a more pronounced maturation of TLOs/MALTs in the LVI-HVI group. It is currently unclear whether these formations play a protective role by contributing to local immunity in chronic inflammatory diseases. However, the structural similarities between TLOs and MALTs and the location of TLOs close to the mucosa suggest that they may perform a similar function, facilitating a local protective response. Nevertheless, further investigations are warranted to assess the cellular and humoral dynamics of these lymphoid organs induced by attenuated isolates.

Intrapancreatic accessory spleens in African swine fever infection of wild boar (Sus scrofa).

Intrapancreatic accessory spleen (IPAS) is one of the most frequent congenital splenic anomalies in humans; however, studies in veterinary medicine are scarce. This study aimed to describe the macroscopic, histopathological and immunohistochemical features of 11 suspected cases of IPAS in wild boar piglets of 3-4 months old. Seven of the 11 animals were immunised with a low virulence isolate of African swine fever virus (ASFV) and subsequently challenged with a highly virulent ASFV isolate (LVI-HVI group). The remaining four animals were exclusively infected with a highly virulent isolate of ASFV (HVI group). Grossly, lesions comprised focal or multifocal reddish areas of variable shape, located on the surface of the pancreatic tail or within the parenchyma. Histological and immunohistochemical studies (anti-CD79 and CD3) confirmed the presence of IPAS in eight of the 11 cases. IPAS shared the same histological structure and alterations as those observed in the original spleen. The immunohistochemical study against ASFV revealed the presence of VP72+ cells in both the spleen and IPAS of seven of the eight piglets. The results of this study describe for the first time the presence of IPAS in ASFV infection of wild boar (Sus scrofa) regardless the isolate and suggest that the infection may induce the development of ectopic splenic tissue due to an increased demand for phagocytic cells from the reticuloendothelial system. However, further studies are needed to understand the immunological mechanisms that trigger the formation of these accessory organs.

Low transmission risk of African swine fever virus between wild boar infected by an attenuated isolate and susceptible domestic pigs.

African swine fever (ASF) is a lethal infectious disease that affects domestic and wild pigs. This complex virus has already affected five continents and more than 70 countries and is considered to be the main threat to the global swine industry. The disease can potentially be transmitted directly through contact with infectious animals, or indirectly by means of contaminated feed or environments. Nevertheless, the knowledge regarding the transmission patterns of different ASF virus isolates at the wildlife-livestock interface is still limited. We have, therefore, assessed the potential transmission of an attenuated ASF virus isolate between infectious wild boar and directly exposed domestic pig. We registered 3,369 interspecific interactions between animals, which were brief and mostly initiated by wild boar. The major patterns observed during the study were head-to-head contact owing to sniffing, thus suggesting a high probability of pathogen transmission. However, only one of the five domestic pigs had a short period of viremia and became serologically positive for ASF virus antibodies. It was additionally discovered that the wild boar did not transmit the virulent virus isolate to the domestic pigs, which suggests that the presence of attenuated ASF virus isolates in affected areas may control the spreading of other more virulent isolates. These outcomes may help make decisions related to large-scale targeted management actions against ASF in field conditions.

Accelerometer-based detection of African swine fever infection in wild boar.

Infectious wildlife diseases that circulate at the interface with domestic animals pose significant threats worldwide and require early detection and warning. Although animal tracking technologies are used to discern behavioural changes, they are rarely used to monitor wildlife diseases. Common disease-induced behavioural changes include reduced activity and lethargy ('sickness behaviour'). Here, we investigated whether accelerometer sensors could detect the onset of African swine fever (ASF), a viral infection that induces high mortality in suids for which no vaccine is currently available. Taking advantage of an experiment designed to test an oral ASF vaccine, we equipped 12 wild boars with an accelerometer tag and quantified how ASF affects their activity pattern and behavioural fingerprint, using overall dynamic body acceleration. Wild boars showed a daily reduction in activity of 10-20% from the healthy to the viremia phase. Using change point statistics and comparing healthy individuals living in semi-free and free-ranging conditions, we show how the onset of disease-induced sickness can be detected and how such early detection could work in natural settings. Timely detection of infection in animals is crucial for disease surveillance and control, and accelerometer technology on sentinel animals provides a viable complementary tool to existing disease management approaches.

A useful tool for the safe diagnosis and control of the two main pandemics of the XXI century: COVID-19 and African Swine Fever disease.

The COVID-19 pandemic and the disease triggered by the African Swine Fever virus are currently two of the main problems regarding public and animal health, respectively. Although vaccination seems to be the ideal tool for controlling these diseases, it has several limitations. Therefore, early detection of the pathogen is critical in order to apply preventive and control measures. Real-time PCR is the main technique used for the detection of both viruses, which requires previous processing of the infectious material. If the potentially infected sample is inactivated at the time of sampling, the diagnosis will be accelerated, impacting positively on the diagnosis and control of the disease. Here, we evaluated the inactivation and preservation properties of a new surfactant liquid for non-invasive and environmental sampling of both viruses. Our results demonstrated that the surfactant liquid effectively inactivates SARS-CoV-2 and African Swine Fever virus in only five minutes, and allows for the preservation of the genetic material for long periods even at high temperatures such as 37°C. Hence, this methodology is a safe and useful tool for recovering SARS-CoV-2 and African Swine Fever virus RNA/DNA from different surfaces and skins, which has significant applied relevance in the surveillance of both diseases.

Safety of African Swine Fever Vaccine Candidate Lv17/WB/Rie1 in Wild Boar: Overdose and Repeated Doses.

African swine fever (ASF) is a highly lethal infectious disease that affects domestic pigs and wild boar. Outbreaks of ASF have grown considerably in the last decade causing important economic consequences for the swine industry. Its control is hampered by the lack of an effective treatment or vaccine. In Europe, the wild boar is a key wild reservoir for ASF. The results of the oral vaccination trial of wild boar with Lv17/WB/Rie1 are hope for this problem. However, this vaccine candidate has certain safety concerns, since it is a naturally attenuated vaccine. Therefore, the current study aims to evaluate the safety of this vaccine candidate in terms of overdose (high dose) and repeated doses (revaccination) in wild boar. Low-dose orally vaccinated animals developed only a slight transient fever after vaccination and revaccination. This was also the case for most of the high-dose vaccinated wild boar, except for one of them which succumbed after revaccination. Although this fatality was related to hierarchical fights between animals, we consider that further studies are required for clarification. Considering these new results and the current epidemiological situation of ASF in wild boar, this vaccine prototype is a promising tool for the control of the disease in these wild populations, although further studies are needed.

A new method for sampling African swine fever virus genome and its inactivation in environmental samples.

African swine fever (ASF) is currently the most dangerous disease for the global pig industry, causing huge economic losses, due to the lack of effective vaccine or treatment. Only the early detection of ASF virus (ASFV) and proper biosecurity measures are effective to reduce the viral expansion. One of the most widely recognized risks as regards the introduction ASFV into a country is infected animals and contaminated livestock vehicles. In order to improve ASF surveillance, we have assessed the capacity for the detection and inactivation of ASFV genome by using Dry-Sponges (3 M) pre-hydrated with a new surfactant liquid. We sampled different surfaces in ASFV-contaminated facilities, including animal skins, and the results were compared to those obtained using a traditional sampling method. The surfactant liquid successfully inactivated the virus, while ASFV DNA was well preserved for the detection. This is an effective method to systematically recover ASFV DNA from different surfaces and skin, which has a key applied relevance in surveillance of vehicles transporting live animals and greatly improves animal welfare. This method provides an important basis for the detection of ASFV genome that can be assessed without the biosafety requirements of a BSL-3 laboratory at least in ASF-affected countries, which may substantially speed up the early detection of the pathogen.

High Doses of Inactivated African Swine Fever Virus Are Safe, but Do Not Confer Protection against a Virulent Challenge.

African swine fever (ASF) is currently the major concern of the global swine industry, as a consequence of which a reconsideration of the containment and prevention measures taken to date is urgently required. A great interest in developing an effective and safe vaccine against ASF virus (ASFV) infection has, therefore, recently appeared. The objective of the present study is to test an inactivated ASFV preparation under a vaccination strategy that has not previously been tested in order to improve its protective effect. The following have been considered: (i) virus inactivation by using a low binary ethyleneimine (BEI) concentration at a low temperature, (ii) the use of new and strong adjuvants; (iii) the use of very high doses (6 × 109 haemadsorption in 50% of infected cultures (HAD50)), and (iv) simultaneous double inoculation by two different routes of administration: intradermal and intramuscular. Five groups of pigs were, therefore, inoculated with BEI- Pol16/DP/OUT21 in different adjuvant formulations, twice with a 4-week interval. Six weeks later, all groups were intramuscularly challenged with 10 HAD50 of the virulent Pol16/DP/OUT21 ASFV isolate. All the animals had clinical signs and pathological findings consistent with ASF. This lack of effectiveness supports the claim that an inactivated virus strategy may not be a viable vaccine option with which to fight ASF.

Distinct African Swine Fever Virus Shedding in Wild Boar Infected with Virulent and Attenuated Isolates.

Since the reappearance of African swine fever virus (ASFV), the disease has spread in an unprecedented animal pandemic in Eurasia. ASF currently constitutes the greatest global problem for the swine industry. The wild boar (Sus scrofa) in which the pathogen has established wild self-sustaining cycles, is a key reservoir for ASFV, signifying that there is an urgent need to develop an effective vaccine against this virus. Current scientific debate addresses whether live attenuated vaccines (LAVs), which have shown promising results in cross-protection of susceptible hosts, may be feasible for vaccinations carried out owing to safety concerns. The objective of this study was, therefore, to compare the ASFV shedding in wild boar infected with virulent and attenuated (LAV) isolates. Different shedding routes (oral fluid and feces) and viremia rates were characterized in wild boar inoculated with Lv17/WB/Rie1 isolate (n = 12) when compared to those inoculated with the virulent Armenia07 isolate (n = 17). In general, fewer animals infected with the Lv17/WB/Rie1 isolate tested positive for ASFV in blood, oral fluid, and feces in comparison to animals infected with the virulent Armenia07 isolate. The shedding patterns were characterized in order to understand the transmission dynamics. This knowledge will help evaluate the shedding of new LAV candidates in wild boar populations, including the comparison with gene deletion mutant LAVs, whose current results are promising.

Clinical Course and Gross Pathological Findings in Wild Boar Infected with a Highly Virulent Strain of African Swine Fever Virus Genotype II.

African swine fever (ASF) is a notifiable disease that in recent years has spread remarkably in Europe and Asia. Eurasian wild boar (Sus scrofa) plays a key role in the maintenance and spread of the pathogen. Here we examined gross pathology of infection in wild boar with a highly virulent, hemadsorbing genotype II ASF virus (ASFV) strain. To this end, six wild boars were intramuscularly inoculated with the 10 HAD50 Arm07 ASFV strain, and 11 wild boars were allowed to come into direct contact with the inoculated animals. No animals survived the infection. Clinical course, gross pathological findings and viral genome quantification by PCR in tissues did not differ between intramuscularly inoculated or contact-infected animals. Postmortem analysis showed enlargement of liver and spleen; serosanguinous effusion in body cavities; and multiple hemorrhages in lungs, endocardium, brain, kidneys, urinary bladder, pancreas, and alimentary system. These results provide detailed insights into the gross pathology of wild boar infected with a highly virulent genotype II ASFV strain. From a didactic point of view, this detailed clinical course and macroscopic description may be essential for early postmortem detection of outbreaks in wild boar in the field and contribute to disease surveillance and prevention efforts.

Adenovirus-vectored African Swine Fever Virus Antigens Cocktail Is Not Protective against Virulent Arm07 Isolate in Eurasian Wild Boar.

: African swine fever (ASF) is a viral disease of domestic and wild suids for which there is currently no vaccine or treatment available. The recent spread of ASF virus (ASFV) through Europe and Asia is causing enormous economic and animal losses. Unfortunately, the measures taken so far are insufficient and an effective vaccine against ASFV needs to be urgently developed. We hypothesized that immunization with a cocktail of thirty-five rationally selected antigens would improve the protective efficacy of subunit vaccine prototypes given that the combination of fewer immunogenic antigens (between 2 and 22) has failed to elicit protective efficacy. To this end, immunogenicity and efficacy of thirty-five adenovirus-vectored ASFV antigens were evaluated in wild boar. The treated animals were divided into different groups to test the use of BioMize adjuvant and different inoculation strategies. Forty-eight days after priming, the nine treated and two control wild boar were challenged with the virulent ASFV Arm07 isolate. All animals showed clinical signs and pathological findings consistent with ASF. This lack of protection is in line with other studies with subunit vaccine prototypes, demonstrating that there is still much room for improvement to obtain an effective subunit ASFV vaccine.